1. Field of the Invention
The present invention provides methods of producing a pro-N-acetylglucosamine-1-phosphodiester xcex1 N-acetyl glucosimanidase (phosphodiester xcex1-GlcNAcase), in mammalian cells deficient in the furin proteolytic enzyme and methods of making lysosomal hydrolases having oligosaccharides modified with N-acetylglucosamine-1-phosphate.
2. Discussion of the Background
Lysosomes are organelles in eukaryotic cells that function in the degradation of macromolecules into component parts that can be reused in biosynthetic pathways or discharged by the cell as waste. Normally, these macromolecules are broken down by enzymes known as lysosomal enzymes or lysosomal hydrolases. However, when a lysosomal enzyme is not present in the lysosome or does not function properly, the enzymes specific macromolecular substrate accumulates in the lysosome as xe2x80x9cstorage materialxe2x80x9d causing a variety of diseases, collectively known as lysosomal storage diseases.
Lysosomal storage diseases can cause chronic illness and death in hundreds of individuals each year. There are approximately 50 known lysosomal storage diseases, e.g., Pompe Disease, Hurler Syndrome, Fabry Disease, Maroteaux-Lamy Syndrome (mucopolysaccharidosis VI), Morquio Syndrome (mucopolysaccharidosis IV), Hunter Syndrome (mucopolysaccharidosis II), Farber Disease, Acid Lipase Deficiency, Krabbe Disease, and Sly Syndrome (mucopolysaccharidosis VII). In each of these diseases, lysosomes are unable to degrade a specific compound or group of compounds because the enzyme that catalyzes a specific degradation reaction is missing from the lysosome, is present in low concentrations in the lysosome, or is present at sufficient concentrations in the lysosome but is not functioning properly.
Lysosomal Storage diseases have been studied extensively and the enzymes (or lack thereof) responsible for particular diseases have been identified (Scriver, Beaudet, Sly, and Vale, eds., The Metabolic Basis of Inherited Disease, 6th Edition, 1989, Lysosomal Enzymes, Part 11, Chapters 61-72, pp. 1565-1839). Within each disease, the severity and the age at which the disease presents may be a function of the amount of residual lysosomal enzyme that exists in the patient.
The lysosomal targeting pathways have been studied extensively and the process by which lysosomal enzymes are synthesized and transported to the lysosome has been well described. Kornfeld, S. (1986). xe2x80x9cTrafficking of lysosomal enzymes in normal and disease states.xe2x80x9d Journal of Clinical Investigation 77: 1-6 and Kornfeld, S. (1990). xe2x80x9cLysosomal enzyme targeting.xe2x80x9d Biochem. Soc. Trans. 18: 367-374. Generally, lysosomal enzymes are synthesized by membrane-bound polysomes in the rough endoplastic reticulum (xe2x80x9cRERxe2x80x9d) along with secretory glycoproteins. In the RER, lysosomal enzymes acquire N-linked oligosaccharides by the en-bloc transfer of a preformed oligosaccharide from dolichol phosphate containing 2 N-acetylglucosamine, 9-mannose and 3-glucose. Glycosylated lysosomal enzymes are then transported to the Golgi apparatus along with secretory proteins. In the cis-Golgi or intermediate compartment lysosomal enzymes are specifically and uniquely modified by the transfer of GlcNAc-phosphate to specific mannoses. In a second step, the GlcNAc is removed thereby exposing the mannose 6-phosphate (xe2x80x9cM6Pxe2x80x9d) targeting determinant. The lysosomal enzymes with the exposed M6P binds to M6P receptors in the trans-Golgi and is transported to the endosome and then to the lysosome. In the lysosome, the phosphates are rapidly removed by lysosomal phosphatases and the mannoses are removed by lysosomal mannosidases (Einstein, R. and Gabel, C. A. (1991). xe2x80x9cCell- and ligand-specific deposphorylation of acid hydrolases: evidence that the mannose 6-phosphate is controlled by compartmentalization.xe2x80x9d Journal of Cell Biology 112: 81-94).
The synthesis of lysosomal enzymes having exposed M6P is catalyzed by two different enzymes, both of which are essential if the synthesis is to occur. The first enzyme is UDP-N-acetylglucosamine: lysosomal enzyme N-Acetylglucosamine-1-phosphotransferase (xe2x80x9cGlcNAc-phosphotransferasexe2x80x9d). GlcNAc-phosphotransferase catalyzes the transfer of N-acetylglucosamine-1-phosphate from UDP-GlcNAc to the 6 position of 1,2-linked mannoses on the lysosomal enzyme. The recognition and addition of N-acetylgluocosamine-1-phosphate to lysosomal hydrolases by GlcNAc-phosphotransferase is the critical and determining step in lysosomal targeting. The second step is catalyzed by N-acetylglucosamine-1-phosphodiester -N-Acetylglucosaminidase (xe2x80x9cphosphodiester xcex1-GlcNAcasexe2x80x9d). Phosphodiester xcex1-GlcNAcase catalyzes the removal of N-Acetylglucosamine from the GlcNAc-phosphate modified lysosomal enzyme to generate a terminal M6P on the lysosomal enzyme.
The present inventors have discovered that the phosphodiester xcex1-GlcNAcase comprises a pro-peptide sequence between the signal sequence and the sequence of the active component of the protein sequence. This pro-peptide sequence is proteolytically cleaved to yield a mature active form of phosphodiester xcex1-GlcNAcase. The activity of uncleaved phosphodiester xcex1-GlcNAcase, i.e., containing the pro-peptide sequence was significantly lower than the activity of the phosphodiester xcex1-GlcNAcase when the pro-peptide sequence was cleaved. The inventors have revealed that the pro-peptide sequence contains a recognition site for the site-specific protease Furin and that Furin mediates cleavage of phosphodiester xcex1-GlcNAcase to it""s mature form.
Based on this finding, the invention provides processes of making lysosomal hydrolase in cells which are deficient in Furin and thus have the uncleaved form of phosphodiester xcex1-GlcNAcase. By making the lysosomal hydrolases in these cells, the lysosomal hydrolase is modified with an N-acetylglucosamine-1-phosphate moiety and is not removed, or removed at a low efficiency. After expression and recovery of the lysosomal hydrolase from these Furin deficient cells, the lysosomal hydrolase can be treated with an active form of phosphodiester xcex1-GlcNAcase thereby removing the N-acetylglucosamine moiety to yield a highly phosphorylated lysosomal enzyme, which can be used in enzyme replacement therapies to treat patients suffering from lysosomal storage diseases.
Thus, the method facilitates a simple and reliable method of producing lysosomal hydrolases with the appropriate phospho-modifications thereby reducing the steps necessary to produce a lysosomal hydrolase for therapeutic or experimental use. Additional advantages include that the N-acetylglucosamine-1-phosphate modified oligosaccharides will not bind to trans-Golgi mannose 6-phosphate receptors resulting in secretion of a greater proportion synthesized lysosomal enzyme thereby improving the yield. Additionally, because less lysosomal enzyme is trafficked to the lysosome there should be less processing to mature forms facilitating the preparation of pure precursor lysosomal enzyme preparations. Oligosaccharides not modified by N-acetylglucosamine-1-phosphate should be processed to complex-type oligosaccharides reducing the number of mannose contained in the lysosomal enzyme thereby reducing affinity for mannose receptors.
Accordingly, an object of the present invention is to provide methods of producing lysosomal hydrolases having an Oligosaccharide modified with N-acetylglucosamine-1-phosphateby expressing a nucleotide sequence encoding the lysosomal hydrolase in a mammalian cell that is deficient in the protease Furin.
Another object of the present invention is methods for producing a phosphodiester xcex1-GlcNAcase having its pro-peptide intact by culturing cells or selecting cells that are furin deficient, where the selection is preferably conducted using Pseudomonas exotoxin A.